Selective involvement of serum response factor in pressure-induced myogenic tone in resistance arteries

OBJECTIVE: In resistance arteries, diameter adjustment in response to pressure changes depends on the vascular cytoskeleton integrity. Serum response factor (SRF) is a dispensable transcription factor for cellular growth, but its role remains unknown in resistance arteries. We hypothesized that SRF is required for appropriate microvascular contraction. METHODS AND RESULTS: We used mice in which SRF was specifically deleted in smooth muscle or endothelial cells, and their control. Myogenic tone and pharmacological contraction was determined in resistance arteries. mRNA and protein expression were assessed by quantitative real-time PCR (qRT-PCR) and Western blot. Actin polymerization was determined by confocal microscopy. Stress-activated channel activity was measured by patch clamp. Myogenic tone developing in response to pressure was dramatically decreased by SRF deletion (5.9+/-2.3%) compared with control (16.3+/-3.2%). This defect was accompanied by decreases in actin polymerization, filamin A, myosin light chain kinase and myosin light chain expression level, and stress-activated channel activity and sensitivity in response to pressure. Contractions induced by phenylephrine or U46619 were not modified, despite a higher sensitivity to p38 blockade; this highlights a compensatory pathway, allowing normal receptor-dependent contraction. CONCLUSIONS: This study shows for the first time that SRF has a major part to play in the control of local blood flow via its central role in pressure-induced myogenic tone in resistance arteries

Abnormal Placental Development and Early Embryonic Lethality in EpCAM-Null Mice

BACKGROUND: EpCAM (CD326) is encoded by the tacstd1 gene and expressed by a variety of normal and malignant epithelial cells and some leukocytes. Results of previous in vitro experiments suggested that EpCAM is an intercellular adhesion molecule. EpCAM has been extensively studied as a potential tumor marker and immunotherapy target, and more recent studies suggest that EpCAM expression may be characteristic of cancer stem cells. METHODOLOGY/PRINCIPAL FINDINGS: To gain insights into EpCAM function in vivo, we generated EpCAM -/- mice utilizing an embryonic stem cell line with a tacstd1 allele that had been disrupted. Gene trapping resulted in a protein comprised of the N-terminus of EpCAM encoded by 2 exons of the tacstd1 gene fused in frame to betageo. EpCAM +/- mice were viable and fertile and exhibited no obvious abnormalities. Examination of EpCAM +/- embryos revealed that betageo was expressed in several epithelial structures including developing ears (otocysts), eyes, branchial arches, gut, apical ectodermal ridges, lungs, pancreas, hair follicles and others. All EpCAM -/- mice died in utero by E12.5, and were small, developmentally delayed, and displayed prominent placental abnormalities. In developing placentas, EpCAM was expressed throughout the labyrinthine layer and by spongiotrophoblasts as well. Placentas of EpCAM -/- embryos were compact, with thin labyrinthine layers lacking prominent vascularity. Parietal trophoblast giant cells were also dramatically reduced in EpCAM -/- placentas. CONCLUSION: EpCAM was required for differentiation or survival of parietal trophoblast giant cells, normal development of the placental labyrinth and establishment of a competent maternal-fetal circulation. The findings in EpCAM-reporter mice suggest involvement of this molecule in development of vital organs including the gut, kidneys, pancreas, lungs, eyes, and limbs

CHAMOMILE PRODUCTION USING SUPPLEMENTARY IRRIGATION AND ORGANIC FERTILIZATION IN SANDY SOILS

The use of medicinal plants in the herbal medicine of Brazilian Health System has intensified production and the need for developing efficient agricultural techniques that promote greater productivity of these species. The objective of this work was to evaluate the chamomile production at field conditions as a function of irrigation depths and organic fertilizer rates. The experiment was conducted in the Universidade do Oeste Paulista (Campus II experimental area), in Presidente Prudente, São Paulo State, Brazil. A triple factorial experimental design was used, consisting of irrigation rates (150, 100, 75, 50, 25 and 0% of the reference evapotranspiration - ETo), organic manure types (poultry and cattle manure) and manure rates (0, 3 and 5 kg m-2), with four replications. The capitula production per plant, capitula dry weight and yield per water input (water use efficiency) were evaluated. The supplementary irrigation combined with organic manure fertilization provided the highest capitula yield for chamomile crop in the Presidente Prudente region. The combination of poultry manure at rate of 5 kg m-2 with water depth equal to 150% of the ETo resulted in higher average values of capitula fresh and dry weight and water use efficiency

Proteomic and Mechanistic Analysis of Spironolactone in Patients at Risk for HF.

OBJECTIVES: This study sought to further understand the mechanisms underlying effect of spironolactone and assessed its impact on multiple plasma protein biomarkers and their respective underlying biologic pathways. BACKGROUND: In addition to their beneficial effects in established heart failure (HF), mineralocorticoid receptor antagonists may act upstream on mechanisms, preventing incident HF. In people at risk for developing HF, the HOMAGE (Heart OMics in AGEing) trial showed that spironolactone treatment could provide antifibrotic and antiremodeling effects, potentially slowing the progression to HF. METHODS: Baseline, 1-month, and 9-month (or last visit) plasma samples of HOMAGE participants were measured for protein biomarkers (n = 276) by using Olink Proseek-Multiplex cardiovascular and inflammation panels (Olink, Uppsala, Sweden). The effect of spironolactone on biomarkers was assessed by analysis of covariance and explored by knowledge-based network analysis. RESULTS: A total of 527 participants were enrolled; 265 were randomized to spironolactone (25 to 50 mg/day) and 262 to standard care ("control"). The median (interquartile range) age was 73 years (69 to 79 years), and 26% were female. Spironolactone reduced biomarkers of collagen metabolism (e.g., COL1A1, MMP-2); brain natriuretic peptide; and biomarkers related to metabolic processes (e.g., PAPPA), inflammation, and thrombosis (e.g., IL17A, VEGF, and urokinase). Spironolactone increased biomarkers that reflect the blockade of the mineralocorticoid receptor (e.g., renin) and increased the levels of adipokines involved in the anti-inflammatory response (e.g., RARRES2) and biomarkers of hemostasis maintenance (e.g., tPA, UPAR), myelosuppressive activity (e.g., CCL16), insulin suppression (e.g., RETN), and inflammatory regulation (e.g., IL-12B). CONCLUSIONS: Proteomic analyses suggest that spironolactone exerts pleiotropic effects including reduction in fibrosis, inflammation, thrombosis, congestion, and vascular function improvement, all of which may mediate cardiovascular protective effects, potentially slowing progression toward heart failure. (HOMAGE [Bioprofiling Response to Mineralocorticoid Receptor Antagonists for the Prevention of Heart Failure]; NCT02556450)

Effects of spironolactone on exercise blood pressure in patients at increased risk of developing heart failure: report from the HOMAGE trial

None of the spironolactone trials in heart failure (HF) assessed the blood pressure (BP) responses to exercise, while conflicting results were reported for exercise capacity. In the HOMAGE trial, 527 patients at increased HF risk were randomized to usual treatment with or without spironolactone (25–50 mg/day). The current substudy included 113 controls and 114 patients assigned spironolactone, who all completed the incremental shuttle walk test at baseline and months 1 and 9. Quality of life (QoL) was assessed by EQ5D questionnaire. Between-group differences (spironolactone minus control [Δs]) were analyzed by repeated measures ANOVA with adjustment for baseline and, if appropriate, additionally for sex, age and body mass index. Δs in the pre-exercise systolic/diastolic BP were −8.00 mm Hg (95% CI, −11.6 to −4.43)/−0.85 mm Hg (−2.96 to 1.26) at month 1 and −9.58 mm Hg (−14.0 to −5.19)/−3.84 mm Hg (−6.22 to −1.47) at month 9. Δs in the post-exercise systolic/diastolic BP were −8.08 mm Hg (−14.2 to −2.01)/−2.07 mm Hg (−5.79 to 1.65) and −13.3 mm Hg (−19.9 to −6.75)/−4.62 mm Hg (−8.07 to −1.17), respectively. For completed shuttles, Δs at months 1 and 9 were 2.15 (−0.10 to 4.40) and 2.49 (−0.79 to 5.67), respectively. Δs in QoL were not significant. The correlations between the exercise-induced BP increases and the number of completed shuttles were similar in both groups. In conclusion, in patients at increased risk of developing HF, spironolactone reduced the pre- and post-exercise BP, but did not improve exercise capacity or QoL. (Figure presented.

I038 Influence des kinines sur les effets cardioet réno-vasculaires de la cyclosporine a

La déficience génétique du Système Kallicréine Kinines (SKK) conduit à un dysfonctionnement artériel, cardiaque et rénal. Des observations faites chez l’homme et l’animal (hypertension, ischémie reperfusion, diabète) ont suggéré qu’une activité réduite du SKK pourrait être la cause de l’induction de ces maladies cardiovasculaires ou participerait à leur progression, alors que certains traitements qui potentialisent les kinines, comme les IEC et la Cyclosporine A (CsA), sont cardioprotecteurs.Le SKK vasculaire aurait donc un rôle protecteur dans les maladies où une vasoconstriction excessive est délétère.ObjectifÉvaluer l’importance du niveau de stimulation du SKK sur le développement d’effets secondaires bénéfiques ou délétères de la CsA.L’administration chronique de l’immunosuppresseur CsA est responsable du développement d’une HTA chez l’homme et le rat. Dans ce modèle murin, la CsA stimule l’activité du SKK, ce qui atténuerait l’augmentation de la PA.Nous déterminerons la capacité de la CsA à modifier l’activité du SKK et évaluerons les effets secondaires liés à un traitement chronique à la CsA en présence (TK+/+) ou absence (Kallicréine Tissulaire Knockout, TK-/-) du SKK.MéthodesTraitement chronique (6 semaines) de souris femelles de 4 mois (TK-/-, n=16 et leurs contrôles TK+/+, n=16) par l’implantation de mini-pompe Alzet 2006 délivrant 30microg/g de souris/jour de CsA ou PBS. Suivis hebdomadaires du poids et de la Pression Artérielle, cage à métabolisme tous les 15 jours et recueil de plasma pour l’évaluation de la fonction rénale. Le sacrifice des animaux permettra la collecte des reins, cœur et gros troncs artériels et leur caractérisation du point de vue moléculaire (RTPCR, HIS, Wblot, et Immunohistologie, caractérisation des SKK, SRA, Endothéline, Vasopressine).RésultatsLes résultats préliminaires de suivis de la PA à 28 jours montrent une augmentation significative de la PA de 15mmHg uniquement chez les TK-/-CsA par rapport aux groupes TK+/+ CsA (112.99±6.83mmHg vs 95.83±4,65mmHg). La comparaison des résultats obtenus pour chacun des groupes étudiés révèlera l’importance du niveau de stimulation du SKK sur le développement de certains mécanismes de cardio- et néphrotoxicité médicamenteuse

N-linked glycosylation of the human bradykinin B2 receptor is required for optimal cell-surface expression and coupling

International audienceTo investigate the glycosylation of the human bradykinin B2 receptor and the functional significance of this modification, we studied receptors mutated at single or multiple combinations of the three potential N-linked glycosylation sites, asparagines N3, N12 and N180, in COS-7, HEK 293 and CHO-K1 cells. Western blot experiments demonstrated that all three extracellular asparagines are glycosylated. The kinetics of bradykinin binding and receptor sequestration remained unchanged after glycosylation had been suppressed. However, the glycosylated receptors were expressed at the cell-surface to a much greater extent than the non-glycosylated receptor and coupling to phospholipase C was less efficient for receptor lacking N-terminal glycosylation. These results indicate that, for the human bradykinin B2 receptor, glycosylation is not required for optimal ligand binding, but plays an important role in cell-surface addressing and receptor function

Receptors for kinins in the human isolated umbilical vein.

1. The human umbilical vein has been found to contract in response to bradykinin (BK) and desArg9BK. 2. The rank order of potency of agonists, in the presence of the B1 receptor antagonist Lys[Leu8]desArg9BK, is as follows: [Hyp3, Tyr(Me)8]BK (pD2 8.88) = [Hyp3]BK (pD2 8.86) = LysBK (pD2 8.81) > or = BK (pD2 8.60) >> [Aib7]BK (pD2 6.38) >> desArg9BK and LysdesArg9BK (inactive). 3. Hoe 140 (pA2 8.42) inhibits the effects of BK while other B2 receptor peptide antagonists are very weak and WIN 64338 is practically inactive. 4. Venoconstrictor responses to desArg9BK of fresh tissues increase with time during the in vitro incubation and reach a maximum after 4-6 h. The activity of Hoe 140 (pA2 5.48) is negligible against B1 receptor agonists. 5. When measured in the presence of the selective B2 receptor antagonist Hoe 140 (400 nM), the order of potency of kinin related peptides on the B1 receptor is Lys[desArg9]BK (pD2 8.60) > desArg9BK (pD2 6.69). BK, LysBK, [Hyp3]BK and other B2 receptor agonists are inactive. 6. The B1 receptor antagonist, Lys[Leu8]desArg9BK (pA2 7.99), inhibits the response of the human vein to B1 receptor agonists (LysdesArg9BK or desArg9BK), but do not alter the effect of BK. 7. The results summarized in this paper indicate that the human isolated umbilical vein is a sensitive preparation containing both B1 and B2 receptors. The human B2 receptor shows some similarity with that of the rabbit (at least for agonist potencies) and differs from the B2 receptor of the guinea-pig. Compared to the rabbit B1 receptor, the human B1 receptor shows low sensitivity to peptides that lack the N-terminal Lys